Tissue Inhibitors of Metalloproteinases (TIMP-l and TIMP-2) regulate the activity of matrix metalloproteinases in the extracellular milieu. Matrix metalloproteinases collectively degrade most if not all matrix macromolecules and appear to be responsible for stromal remodeling in many normal and pathological events. The inhibitors function at two important levels: by blocking activation of the latent matrix metalloproteinase precursors and by forming inhibited complexes with the activated forms of the enzymes. We wish to understand the molecular basis for these functions and to examine how TIMPs regulate extracellular matrix degradation by live cells. In Aim 1, we propose to identify those domains and subdomains of TIMP-1 and TIMP-2 that are important to inhibitory function by peptide- and antibody- competition approaches and by site-directed mutagenesis of sequences targeted by initial competition experiments. We also propose to identify those domains of TIMP-1 and TIMP-2 that are important to forming complexes with the Mr 72K GL and Mr 92K GL zymogens, and to understand the basis for these highly specific interactions. In Aim 2, we propose to examine how TIMPs maintain latency of MMP precursors. We have formulated the novel hypothesis that TIMPs form zymogen complexes with all MMP precursors and that they regulate the rate of opening of the Cys-Zn++- switch which is a prequisite for MMP activation. We will specifically quantify the extent to which TIMPs modify the rate of switch-opening and we will seek to demonstrate formation of dissociable complexes between fibroblast-type collagenase and stromelysin-1 and TIMPs. In Aim 3, the role of TlMPs in regulating the degradation of collagen fibrils by live cells will be analyzed. Two cell based systems will be used, one in which collagen breakdown is naturally blocked because of coexpression of TIMPs and one in which rapid collagen breakdown is induced by addition of plasminogen. The function of specific mutant and wildtype TIMPs in controlling collagen degradation in the plasminogen-supplemented system will be examined by addition or overexpression of these TIMPs by means of a vaccinia virus system. In addition, we will seek to establish that collagen breakdown is blocked in the absence of plasminogen because of endogenous AMP expression. Our approach will be to downregulate either the expression (antisense oligonucleotides or RNA) or the activity (neutralizing peptides antibodies) of one or both TIMPs and to monitor the effect on collagen degradation. The proposed studies will yield a better understanding of the natural control and regulation mechanisms that serve to limit tissue destruction during normal and pathological developments.